Basic Vector Information
Gene/Insert name FnCpf1 (humanized) Alt name type V CRISPR-associated protein Cpf1 (humanized) Alt name Cpf1 (humanized) Alt name Francisella tularensis subsp. novicida U112 Cpf1 (humanized) Species Francisella tularensis subsp. novicida U112 Insert Size (bp) 3910 Promoter T7 Tags / Fusion Proteins 6xHis (N terminal on backbone) MBP (N terminal on insert) TEV site (N terminal on insert) Nucleoplasmin NLS (C terminal on insert) 3xHA (C terminal on insert)
| Basic Vector Information | |||
|---|---|---|---|
| Vector Name | 6-His-MBP-TEV-FnCpf1 | Antibiotic Resistance | Ampicillin |
| Length | 10685 bp | Type | CRISPR Plasmids |
| Source | Feng Zhang | Copy Number | High copy number |
| Promoter | T7 | Cloning Method | Enzyme Cut |
| 5' Primer | T7 promoter | 3' Primer | T7 terminator |
| Fusion Tag | MBP | Expression Method | IPTG induced |
6-His-MBP-TEV-FnCpf1 vector Vector Map
Plasmid Resuspension protocol:
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5.Store the plasmid at -20 ℃.
6-His-MBP-TEV-FnCpf1 vector Sequence
LOCUS Exported 10685 bp ds-DNA circular SYN 04-JUL-2018
DEFINITION Bacterial expression plasmid for protein purification.
ACCESSION .
VERSION .
KEYWORDS 6-His-MBP-TEV-FnCpf1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10685)
AUTHORS Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS,
Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin
EV, Zhang F
TITLE Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas
System.
JOURNAL Cell. 2015 Sep 23. pii: S0092-8674(15)01200-3. doi:
10.1016/j.cell.2015.09.038.
PUBMED 26422227
REFERENCE 2 (bases 1 to 10685)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..10685
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin 532..1120
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature 1306..1445
/note="bom"
/note="basis of mobility region from pBR322"
CDS complement(1547..1738)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/note="rop"
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(2547..3629)
/codon_start=1
/gene="lacI"
/product="lac repressor"
/note="lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
promoter complement(3630..3707)
/gene="lacI"
/note="lacI promoter"
promoter 4016..4034
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 4035..4059
/bound_moiety="lac repressor encoded by lacI"
/note="lac operator"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
RBS 4074..4096
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 4104..9326
/codon_start=1
/note="6His-MBP-TEV-FnCpf1-NLS-3xHA"
/translation="MGHHHHHHKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTV
EHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDA
VRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPY
FTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAE
AAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNK
ELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPN
IPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTGASTGSLVPRGSASENLYFQGMSIY
QEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIE
EILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNL
FNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKG
FHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAE
ELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKG
INEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQ
IAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAV
LEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFE
EILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNN
LLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDE
KFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEG
YKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEF
NIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISE
SYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAEL
FYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFK
SSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDR
MKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFED
LNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKK
MGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFE
FSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIE
YGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDS
RQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNNKR
PAATKKAGQAKKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYA"
CDS 4110..4127
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
regulatory 5082..5091
/regulatory_class="other"
/note="Kozak sequence"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 5244..5261
/codon_start=1
/product="thrombin recognition and cleavage site"
/note="thrombin site"
/translation="LVPRGS"
CDS 5268..5288
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/note="TEV site"
/translation="ENLYFQG"
CDS 9189..9236
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/note="nucleoplasmin NLS"
/translation="KRPAATKKAGQAKKKK"
CDS 9243..9323
/codon_start=1
/product="three tandem HA epitope tags"
/note="3xHA"
/translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
CDS 9348..9371
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/note="FLAG"
/translation="DYKDDDDK"
CDS 9372..9416
/codon_start=1
/product="peptide tag that allows for enzymatic
biotinylation"
/note="AviTag(TM)"
/translation="GLNDIFEAQKIEWHE"
CDS 9432..9449
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
terminator 9516..9563
/note="T7 terminator"
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 9600..10055
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 10081..10185
/gene="bla"
/note="AmpR promoter"
CDS 10186..361
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
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